Analysis of Natural and Synthetic Estrogens at Sub-ppt Levels in Surface and Crude Influent Water with 2D UPLC-MS/MS

Scottish Water provides 1.34 billion liters of drinking water every day and takes away 847 million liters of waste water daily. Answerable to both the Scottish Parliament and the people of Scotland, Scottish Water recognizes that innovation is a key enabler for them to deliver sustainable, high-quality water treatment services.

One of the many compounds they monitor for is the drug class of estrogens. Estrogens are routinely used either as contraceptive medicines or in hormone replacement therapy and can enter aquatic environments via the discharge of final effluent waters. Once waste water is released into the environment, these synthetic hormones can have negative effects on wildlife. Estrogens are believed to have a negative effect on aquatic environments by disrupting the hormonal systems of fish; for example, they may contribute to intersex changes in fish.

In the EU directive 2013/39/EU, 15 priority substances were added to the European Water Framework Directive (WFD, 2000/60/EC). In this update, 17α-ethinyl-estradiol (EE2) and 17β-estradiol were not included in the priority list, but were instead added to a watch list in order to gather further data regarding the presence of these compounds in aquatic environments and the risks they pose.

“Even low concentrations of EE2 have an impact on fish – both their behaviour and their genetics. We have seen a change in the genetic balance in fish, and that they have a harder time catching food. Previous studies have shown that the fish also develop problems with procreation. This can lead to the complete disappearance of an entire fish population, and consequences for entire ecosystems,” said Lina Nikoleris at Lund University in Sweden in a 2016 paper.

Waters partnered with Scottish Waters to develop and validate an analytical method for the analysis of 17β-estradiol, estrone, and 17α-ethinylestradiol in surface water, crude influent, and final effluent from a waste water treatment plant utilizing off-line solid phase extraction followed by analysis on an ACQUITY UPLC System with 2D LC Technology, coupled to a Xevo TQ-S Mass Spectrometer.

Analytical goal: Confirm and quantify the presence of natural and synthetic estrogens in surface and final effluent waters at sub-part-per-trillion (ppt) levels.

The combination of off-line SPE followed by analysis on the ACQUITY UPLC System with 2D LC Technology and Xevo TQ-S for MS/MS quantification allows for ultra-sensitive detection of natural and synthetic estrogens in surface and treated water.

Concentration with sample prep

Surface water samples were initially extracted utilizing an optimized method on an off-line Oasis HLB Solid Phase Extraction (SPE) Cartridge. Crude influent and final effluent samples were first filtered, and then underwent the same Oasis HLB offline extraction step. This was followed by a second SPE step utilizing Sep-Pak Silica Cartridges.

Additional concentration with on-line 2D LC

Extracted samples were injected onto the ACQUITY UPLC System with 2D LC Technology, coupled to the Xevo TQ-S tandem (triple) quadrupole mass spectrometer. The use of UPLC in a two-dimensional chromatographic separation allows for a further concentration of the sample on-line by utilizing a Direct Connect Oasis HLB Column. Once concentrated on the on-line column the compounds were then eluted onto an ACQUITY UPLC BEH C18 Column to provide a reversed-phase separation. Figure 1 provides an example of the 2D chromatography achieved.

The samples were determined using electrospray in negative ion mode allowing two MRM transitions per compound in the quantitative MS/MS method. The unique RADAR function of the Xevo TQ-S mass spec, which allows simultaneous acquisition of both MRM and full scan MS data, was also employed to aid with method development.

Validation

This method has undergone a full validation and was found to meet the required performance criteria, providing excellent linearity, as shown in Figure 2, and precision for this challenging analysis. A chromatogram showing detection of 17β-estradiol in low level final effluent spiked sample (0.6 ng/L) is shown in Figure 3.

The performance test data was comprised of a a NS30-style set (NS30, 1989) of tests of 11 batches of duplicate analyses of blanks, low and high standards, and low and high spiked samples of effluent. Spiked recovery data was also produced for river and influent matrices.